Regulating intracellular protein flux is a critical component of cellular metabolism and defects in this process are linked to multiple human diseases including neurodegeneration, cystic fibrosis, and cancers. In eukaryotes, the 20S proteasome performs the bulk of proteolysis in the cytosol and nucleus and is vital in diverse processes such as protein quality control, signal transduction, cell cycle progression, DNA repair, and antigen presentation. The two-fold symmetric eukaryotic 20S is composed of 28 (14 x 2) subunits that form a ~730 kDa. barrel-like structure, which contains the six proteolytic sites. The complicated architecture renders eukaryotic 20S incapable of self-assembly. Recently, a heterodimeric Proteasome Associated Biogenesis factor (Pba1/2 in yeast; PAC1/2 in mammals) has been discovered that acts as a 20S assembly chaperone. Interestingly, Pba1/2 also contains a C-terminal HbYX sequence, which is an established 20S activating motif, and our preliminary data show that Pba1/2 binds to the assembled 20S proteasome. Our primary objective is to determine the structural basis for the 20S-Pba1/2 interaction, and toward this goal we have crystallized a Pba1/2-20S complex and collected X-ray diffraction data to 3.0E resolution. We will use the forthcoming structural information to guide genetic and biochemical investigations into the functional importance of Pba1/2-20S interactions in yeast. We will also determine if Pba1/2 interacts with 20S assembly intermediates, such as subsets of 1-subunits and other chaperones. Successful outcome will provide structural and functional insight into proteasome function in general and Pba1/2 in particular.